p > .20

p>.20

p > .20

Normality

p <.01

LogNormality

fablel Abreviations (code), descriptions and results of the normality tests (Lillie fors) for quantitative parameters in the characterization attic Las Lomas rabbit population

g/100 gIIOO

Alpha-1 -globulin

Alpha-2-globulin

13GA1

BCA2

Triglyceride

BCO1=

BTGL

number of processed individuals,

M-

Cholesterol

BAGR

M --

11100 mgl°o

Albumine-globulin ratio

BPR

43

43

28

28

28

28

28

28

28

14

24

49.0

37.9

1.82

6.65

.856

.483

.388

.349

4- 26

6.07

355.2

mean', Std - standard deviation, 1)% = Sal/M.

mg"

g110)

Total proteins

BGG

W

g/100

Beta-globulin

Gamma-globulin

BGB

11100

Albumin

g1100

000,/p1

BALB

Serum biochemistry (B)

Iced blood cells

HR

1 000/pi

Fi

Platelets

IiPT

22.0

24

Meaucell hemoglubine

lI MC

62.8

36.9

13.1

8.70

22

3.78

25

24

Fl

I1einatocyte

Mean corpuscttle volume

[(CT

HMV

Hemoglobin

1GB

23

1 OOOIN!

33

25

While blood cells

HW

%

39

°/a

Losinopbiles

HE.U

%

Will

Monocytes

HMO

.30

27.13

16.61

55.4

43.8

16.6

10.9

319

.73

28.9

29

42.5

43.5

10.3

16.9

56.8

9.0

12.2

10.1

15.0

38.5

186.4

659

.23

.1 6

.15

.44

1_03

201.68

1.99

7.68

3.73

1.96

3.35

.41

2.49

p > .20

p ~ .20

p > .20

p > .20

p > .20

p<.01

.15 < p <.20

.OI
p>.20

p > .20

p> .20

.10
p>.20

p> ,20

p>.20

p> 20

p<.01

p<_0I

p<.01

p>.20

.05
.10
588

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Fig. I. Principal components analysis of 6 rabbit skull measurements (see Table l) (30 male and female adults) from Las Lomas (FixF2 projections for variables and individuals)

33. DISCRETE QUALITATIVE CHARACTERS

Eight original non-metrical discrete osteological variables were noted (Fig. 2): -- the shape of the " scutellum" of the supraoccipital (DSO, square (DS la) or trapezoidal (DSIb); - in norma dorsalis, the shape of the lateral edges of crista nuchae (DS2), straight (DS2a) or biconcave (DS2b); - the shape of the aboral edge of crista nuchae ( DS3), on the protuberantia occipitalis externa, can be concave (or notched) on the sagittal line (DS3a), wide accolade bracket shaped (DS3b) or open-V shaped, pointing backwards (DS3c); -- the dorsal outline of the foramen magnum (DS4) displays five types (DS4d being absent in the sample from Las Lomas); - the bully tympanica (DS5), smooth and rounded (DS5a), with an oblique ridge (DSSb), with two weak parallel folds (DS5c) or with a central hump more or less protruded (DS5d); - the second sacral vertebra can participate in the articular surface (DSAb) or not (DSAa); - the lateral trochanteric crest of the femur, notched in the middle (DFa) or unbroken (DFb);

Multipe characterization of a population of rabbits

Fig. 2 Description of osteological discrete characters (D) used in characterization of rabbits: skull (DS). sacrum (OSa), femur (DF), tibia (DT)

589

590

Vigne et al.

- the distal protuberence of the tibial crest, rounded (DTa), protruded with ( DTb), or without (DTc) a protruded notch. Depending on the character, less than 10% of bones could not be classified ( DSn?; Table 2) except for DSF (11.4%); we can therefore consider that they are nearly all discrete. None are sex-correlated. A number of types observed at Las Lomas are significantly absent in some of the other populations (in comparison to the Las Lomas samples: e test): DFb and DTa in northern France wild rabbits; DS4a and DFa in - domestic rabbits. DS4d seems to be absent from the Las Lomas and Kerguelen populations. Finally, in the Las Lomas population, the percentages of individuals are never equally distributed between the different types of each character (Table 2). and a large number of distributions undergo significant changes according to populations (Callon and Vigne, unpublished data). Combinations of the three discrete characters of the acrocranial part of skull (DS1. DS2. DS3) also yield useful possibilities for characterization (Table 3): three combinations (DSla-DS2b-DS3b, DSIb-DS2b-DS3c and, especially DSIa-DS2b--DS3a) are shown by more than 10% of the Las Lomas rabbits, whilst others are very rare. In addition, two combinations (DSIa-DS2b-DS3 and DSla-DS2b-DS3b) seem to be absent at Las Lomas. yet present in two of the other samples under consideration: three of the ten Las Lomas combinations are significantly absent from one of the four compared populations ( DS I a-DS2a-DS3a, DS 1 a-DS2b-DS3a and DSIb-DS2a-DS3a). Although it is necessary to test these characters on a larger number of rabbit populations, the presence/absence and the frequencies of these osteological discrete characters and their combinations, seem to be useful tools in morphologically characterising rabbit populations. 3.4. mtD*4A

Every individual so far analysed displays heteroplasmy for its mtDNA, a peculiar property shared by other Leporidae and described previously (Ennafaa et ai. 1987, Biju-Duval et al. 1991). The 14 restriction endonucleases recognized 53 sites along the different molecules (Fig. 3A), 8 of them being polymorphic (i.e. do not exist in all individual extracts). The repartition of the polymorphic sites leads to the description of 7 different mtDNA types named A I to A7 (Fig, 3B). These mtDNA types can be organised in a parsimonious network (Fig. 4), Most of the steps from a given type to the closest one involve only one mutational event, detected through loss or gain of one site, and probably due to nucleotide substitution. In one case (A2 to A6 or reverse), three events are required. From the overall data, nucleotide diversity is estimated at 0,3%. Taking into - account a divergence rate of 2% per Myrs (13 row n, George and W i [so n i979; Wilson et al. 1985), the molecule ancestor of all mtDNA types present in the Las Lomas population may be traced back 150 000 years ago.

Multipe characterization of a population of rabbits

591

Tablet Percentages of osteological discrete morphotypes (see Fig 2) For Las Lomas rabbits (LL) and presence {*) of these morphotypes in other comparison rabbit populations

W - French wild rabbits: Z - Zernbra Island: K - Kerguelen island; I) - French domestic rabbits. Heavy type indicates significant differences le rest) within the LL sample. Frames indicate significant absences in other populations by reference to LL frequencies

592

l--D. Vigne et al.

Table3 a- percentages of combinations of acrocranial discrete characters (see Figure 2) in Las Lomas rabbits (N = 34: only 26.5% is significantly different from 5 . 9%; e test); b- comparison of rabbit populations within which these combinations have not been observed (abbreviations: see Figure 2); bold type indicates significant absences by reference to LL frequencies (e test) a

DS2a DS2b DS2a DS2b

DSla DS1b

b DSIa DSIb

DS3a

DS3b

8.8 26.5 5.9 .0

5.9 11.8 5,9 .0

DS3a

DS3b

DS3c

IC Z D

Z Z

Z

r

DS2a DS2b DS2a _DS2b

K

D

DS3c I

I

I

5.9 8.8 8.8 11.8

Z. K

4. DISCUSSION AND CONCLUSION 41. VALUE AND BIOLOGICAL MEANING OF THESE VARIABLES FOR EXTANT POPULATIONS

The biological meaning of the variables used in characterizing the Las Lomas population differs for each. - Strong polymorphism in the mtDNA reflects mutation events in different female lineages descended from an ancestral molecule, probably with no selective pressure. - Osteological discrete characters should reflect nuclear genes that might have been selected by more or less ancient environmental pressures. - Blood variables are more or less determined at the genetic level (i mmunoglobulins, cholesterol etc.), but we cannot distinguish the genetic contribution from the environmental one; the environmental source of variation (seasons, photoperiod etc.) has been reduced by sampling the rabbits in a very short period and in a very small plot. -- Body and skeleton sizes may also be related to nuclear genes, but they are much more highly dependant on environmental changes. It appears that the range of variables cannot be compared with each other. The present set of variables, however, yields very different and complementary investigatory approaches which are able to reflect as widely as possible, heterogeneity and specific characteristics of rabbit populations. The process used for the Las Lomas rabbits can be taken as an example, but it must be repeated on many other selected populations for a full phylogeographic reconstruction.

Multipe characterization of a population of rabbits

593

Fig. 3. MtDNA restriction map (A) and description of the seven mtDNA types (AI to A7) in the Las Lomas rabbit sample (B). Letters a to h indicate polymorphic sites. The map is constructed on the basis of a virtual middle-sized molecule (17400 bp), mtDNA loop is linearised by opening on an Avail site, probably common to man, mouse and cow, very close to the beginning of the coding region. Circles indicate changing size fragments (heteroplasmy)

594

J.-D.

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Fig. 4. Network for the 7 Las Lomas rabbit mitotypes AI to A7. For polymorphic sites a to h. see Fig. 3. Each dash between mtDNA types indicates one mutational event.

4.2. USE OF SELECTED VARIABLES FOR AN HISTORICAL APPROACH

Some of the variables in the above set can be estimated in the fossil samples, such as mtDNA polymorphism (Hardy et ai. 1994), osteological discrete fossil characters and measurements. Only the third have been applied to "populations" in the vicinity of Las Lomas. Numerous osteometrical data indicated that the size of rabbits from the western lowlands of Andalusia did not change significantly between the Neolithic, Bronze Age and Classical Antiquity, but was smaller on coastal plains than inland (M i 1 z 1986). Measurements from Cerro de la Virgen (CV), in the interior (Galera, Prov. Granada; D r i e s c h and B a e s s n e c k 1970), offers the largest reference sample of ancient Andalusian rabbits (Table 4). There are only two archaeozoological skull measurements (S7, S9), but these skulls always appear slightly larger (e = 3.3 for CV) than those from Las Lomas. The only exception is with Bronze Age rabbits from Fuente Alamo (FA; Driesch et al. 1985), whose skulls were significantly smaller than those at Las Lomas (e = 3.5). All postcranial measurements, either from the interior (CV; Table 4) or peripheral ancient populations (FA) are much larger than those of Las Lomas (11 < e < 40). These comparisons indicate a strong allometric decrease in rabbit size in southern Spain during the last 2000 yrs. Skull size has not decreased (or only

Multipe characterization of a population of rabbits

595

slightly), but limb bones were shortened by 10 to 15% in comparison to fossil bones from Cerro de la Virgen. The decrease in size indicates that the Las Lomas population has undergone strong environmental and possibly nuclear genetic changes during the last 2000 yrs. Nevertheless, the range of each variable does not seem to have been reduced, including mtDNA, which means that the population has not undergone any "bottleneck" effect during this time. Table4 Dsteometric comparison of 5 measurements (L.1. SH. SU. SF, ST in mm: see Table 1) between present day Las Lomas rabbits ILL). and Late Neolithic to Bronze Age rabbits from Cerro de la Virgen(CV; Driesch and Boessneck 1970) LJ(=S9)

N M Std

i

St-1

1

SU

LL

CV

LL

CV

LL

CV

36

77

34

100

35

23

13.8

14.2

53.9

60.6

70.6

0.50

N - number of

1

0.76

1.71

61.5 2.26

2.05

2.87

}

I

SF

ST

LL

CV

LL

CV

36

96

36

214

68.6

77.9

80.0

1.81

2,55

2.23

94.1

I

1.96

measurements; M -- mean; Std - standard deviation

5. REFERENCES C. Ennafaa H.. Dennebouy N, Monnerot M. Mignotte F., PDF 13iju-Duval Soriguer R. C.. El,Gaa'ied A.. El Hill A. and Mounolou J.-C. 1991 Mitochondrial DNA evolution in lagomorphs: origin of systematic beteroplasmy and organization of diversity in European rabbits - .1. Mol. Evol. 33: 92-102. Brown G. G.. George M. and Wilson A. C. 1979 - Rapid evolution of mitochondrial DNA - Proc. Natl. Acad. Sci. USA 76: 1967-1971. Cabrera A. 1923 - Sabre los conejos de Marruecos - Bol. Real Soc. Esp. H.M. 23: 356-368. Donna rd E. I980 - Recherches sur les Ieporines quaternaires (Pleistocene rnoyen at superieur. Holocene) - These 3e cycle Univ. Bordeaux 1 (n° 1764). D t i e se h A. von den 1976 - A guide to the Measurement of Animal Bones from Archaeolog ical Sites - Peabody Mus. Bull.. Harvard Univ. I: 1-137. D r i e s c h A. von den and B o e s s n e c k J. 1970 - Vorgeschichtliche Kaninchen aus zwei sildspanischen Siedluegen - Saugetierk. Mitteil. 13(2): 127-151. D r i e s c h A. von den. 13 o e s s n e c k J.. Kokabi M. and Schaffer I. 1985 - Tierknochen aus der Bronzezeitlichen hehensiedlungen Fuente Alamo. Provinz Almeria - Studien Ober Friihe Tierknochenfunde v. der lberischen Flaibinsel. 9: 1-75. Ennafaa H.. Monnerot M.. El Gaa'ied A. and Mounolou J.-C. 1987 - Rabbit mitochondrial DNA: preliminary comparison between some domestic and wild animals -Genet. Sef. Evol. 19 (3): 279-288. F e r r a n d N. 1989 -- Biochemical and Genetic Studies on Rabbit Hemoglobin. I. Electrophoretic Polymorphism of the 13 Chain - Biochemical Genetics 27 (II-12): 673-677.

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F c r r a n d N. 1990 - Biochemical and Genetic Studies on Rabbit Hemoglobin. Il. Electrophoretic Polymorphism a Chain - Biochemical Genetics 28 (3-4). F e r r a n d N. and A m o r i m A. 1990 - Genetic Polymorphism of b -- Aminolevulinic Acid Dehydratase (E.C.4.2. 1.24. Alad) in the Domestic Rabbit - Animal Genetics 21. F e r r a n d N., C a r v a i h o G. and A m o t i m A. 1988 - Transferin (Tf) polymorphism in wild rabbit, Orvcrolagus cuniculus - Animal Genetics 19: 295-300. Hardy C o Vigne J.-D., Casares D., Denne bony N., Mounolou J.-C. and M o n n e r o t M. 1994 - Origin of European Rabbit ( Oryctalagus cuniculus) in a Mediterranean island: zooarchaeology and ancient DNA examination - J. Evol. Biol. 7: 217-226. M i l z H. 1986 - Die Tierknochenfunde aus drei argarzeitlichen Siedlungen in der Provinz Granada (Spanien) - Studien fiber friihe Tierknochenfunde v. der iherischen Halbinsel, 10: 78-86. N e i M. and L. i W H. 1979 - Mathematical model for studying variation in terms of restriction endonucleases - Proc. Natl. Acad. Sci. USA 76: 5269-5273. S o r i g u e r R. 1980 - El conejo. Qryctalagus cuniculus (L), en Andalucia occidental: parametros corporales y curva de crecimiento - Doaana Acta Verteb. 7(1); 83-90. Van d e r Loo W.. F e r r a n d N. and S o r i g u e r R. C. 1991 - Estimation of gene diversity at the b-locus of the constant region of the immunoglobulin light chain in natural populations of European rabbit (Oryctolagus cuniculus) in Portugal. Andalusia and on the Azorean Islands - Genetics 127: 789-799. Wilson A. C., Cann R. L., Carr S., George M., Gyllenstein U. B., Helm-Bychowsky K. M., Higuchi It. G.. Palumbi S. R.. Prager E. M., S a g e E. D. and S t o n e k i rig M. 1985 - Mitochondria) DNA and two perspectives on evolutionary genetics - Biol. J. Linn. Soc. 26: 375-400.